Abstract：
Genome editing of allogeneic T cells can provide \"off-the-shelf\" alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator-like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3\' long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 106 to 2.0 × 106 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.
摘要：
同种异体T细胞的基因组编辑可以为自体嵌合抗原受体（CAR）T细胞疗法提供“现成的”替代方案。先前已经使用转录激活因子样效应核酸酶（TALEN）介导的敲除研究了在阿仑单抗存在下破坏T细胞受体α链（TRAC）以防止移植物抗宿主病（GVHD）和去除CD52（分化簇52）以获得生存优势。这里,我们通过在CAR19慢病毒载体的3'长末端重复序列中掺入自我复制的CRISPR指导RNA表达盒,将下一代CRISPR-Cas9编辑和CAR表达链接应用于TRAC和CD52的多重DNA编辑.产生并冷冻保存TT52CAR19T细胞的三个细胞库。第一阶段，开放标签，我们进行了非随机临床试验,对6例复发/难治性CD19阳性B细胞急性淋巴细胞白血病(B-ALL)患儿进行了治疗(NCT04557436).淋巴耗竭包括氟达拉滨，环磷酰胺,和阿仑珠单抗，然后单次输注每公斤0.8×106至2.0×106个CAR19T细胞，无即时毒性。6名输注TT52CAR19T细胞的患者中有4名表现出细胞扩增，实现流式细胞术缓解，然后接受异基因干细胞移植。两名患者需要II级细胞因子释放综合征的生物干预，一名患者出现短暂的IV级神经毒性,一名患者出现皮肤GVHD,在移植调理后解决了。其他并发症在预期之内，并达到了主要安全目标。本研究提供了可行性的论证，安全,和CRISPR工程免疫疗法的治疗潜力。
